The proline-activating activity of the multienzyme gramicidin S synthetase 2 can be recovered on a 115-kDa tryptic fragment.
The multienzyme gramicidin S synthetase 2 was treated with trypsin to obtain fragments capable of activating proline. Three different active fragments were detected. The course of proteolysis was simulated by using a concentration range of trypsin; the cleavage pattern indicated that one of the fragments was particularly stable. This fragment was purified and shown to have a molecular mass of 115 kDa. It was compared chromatographically, by SDS/PAGE, and enzymatically to a Pro-activating fragment produced by a gramicidin-S-negative mutant. It can be concluded that the proteolytic fragment represents a structure which is contained on a continuous part of the polypeptide chain of gramicidin S synthetase 2 and has a relatively compact structure. This provides evidence that the multienzyme gramicidin S synthetase 2 is, at least in part, constructed from functional domains. An approach towards extending these studies to other parts of the gramicidin S synthetase 2 molecule has also been devised. This work complements recombinant DNA studies in the area, providing stable functional fragments.
Skarpeid
H J
HJ
Biokjemisk Institutt, University of Oslo, Norway.
Zimmer
T L
TL
Shen
B
B
von Döhren
H
H
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000591:Amino Acid Isomerases / Q000302:isolation & purification*; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D001711:Biotransformation / Q000187:drug effects; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D004789:Enzyme Activation; D015151:Immunoblotting; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000502:physiology; D010453:Peptide Synthases / Q000302:isolation & purification*; D011392:Proline / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D011392:Proline; D014357:Trypsin; D000591:Amino Acid Isomerases; D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2